Identification of functional open reading frames in chloroplast genomes

We have used a rapid computer dot-matrix comparison method to identify all DNA regions which have been evolutionarily conserved between the completely sequenced chloroplast genomes of tobacco and a liverwort. Analysis of these regions reveals 74 homologous open reading frames (ORFs) which have been conserved as to length and amino acid sequence; these ORFs also have an excess of nucleotide substitutions at silent sites of codons. Since the nonfunctional parts of these genomes have become saturated with mutations and show no sequence similarity whatsoever, the homologous ORFs are almost certainly functional. A further four pairs of ORFs show homology limited to only a short part of their putative gene products. Amino acid sequence identities range between 50 and 99%; some chloroplast proteins are seen to be among the most slowly evolving of all known proteins. A search of the nucleotide and amino acid sequence databanks has revealed several previously unidentified genes in chloroplast sequences from other species, but no new homologies to prokaryotic genes.     

The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals

Summary The entire chloroplast genome of the monocot rice (Oryza sativa) has been sequenced and comprises 134525 bp. Predicted genes have been identified along with open reading frames (ORFs) conserved between rice and the previously sequenced chloroplast genomes, a dicot, tobacco (Nicotiana tabacum), and a liverwort (Marchantia polymorpha). The same complement of 30 tRNA and 4 rRNA genes has been conserved between rice and tobacco. Most ORFs extensively conserved betweenN. tabacum andM. polymorpha are also conserved intact in rice. However, several such ORFs are entirely absent in rice, or present only in severely truncated form. Structural changes are also apparent in the genome relative to tobacco. The inverted repeats, characteristic of chloroplast genome structure, have expanded outward to include several genes present only once per genome in tobacco and liverwort and the large single copy region has undergone a series of inversions which predate the divergence of the cereals. A chimeric tRNA pseudogene overlaps an apparent endpoint of the largest inversion, and a model invoking illegitimate recombination between tRNA genes is proposed which accounts simultaneously for the origin of this pseudogene, the large inversion and the creation of repeated sequences near the inversion endpoints.     

Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages

In the plant chloroplast genome the codon usage of the highly expressed psbA gene is unique and is adapted to the tRNA population, probably due to selection for translation efficiency. In this study the role of selection on codon usage in each of the fully sequenced chloroplast genomes, in addition to Chlamydomonas reinhardtii, is investigated by measuring adaptation to this pattern of codon usage. A method is developed which tests selection on each gene individually by constructing sequences with the same amino acid composition as the gene and randomly assigning codons based on the nucleotide composition of noncoding regions of that genome. The codon bias of the actual gene is then compared to a distribution of random sequences. The data indicate that within the algae selection is strong in Cyanophora paradoxa, affecting a majority of genes, of intermediate intensity in Odontella sinensis, and weaker in Porphyra purpurea and Euglena gracilis. In the plants, selection is found to be quite weak in Pinus thunbergii and the angiosperms but there is evidence that an intermediate level of selection exists in the liverwort Marchantia polymorpha. The role of selection is then further investigated in two comparative studies. It is shown that average relative codon bias is correlated with expression level and that, despite saturation levels of substitution, there is a strong correlation among the algae genomes in the degree of codon bias of homologous genes. All of these data indicate that selection for translation efficiency plays a significant role in determining the codon bias of chloroplast genes but that it acts with different intensities in different lineages. In general it is stronger in the algae than the higher plants, but within the algae Euglena is found to have several unusual features which are noted. The factors that might be responsible for this variation in intensity among the various genomes are discussed. Received: 6 June 1997 / Accepted: 24 July 1997     

Using bacteria to analyze sequences involved in chloroplast gene expression

The complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. Other genes involved in photosynthesis, respiratory electron transport, and membrane-associated transport in chloroplasts were predicted by the amino acid sequence homology and secondary structure of gene products. Thirty-three open reading frames in the liverwort chloroplast genome remain unidentified. However, most of these open reading frames are also conserved in the chloroplast genomes of two species, a liverwort, Marchantia polymorpha, and tobacco, Nicotiana tabacum, indicating their active functions in chloroplasts.Abbreviations bp base pair - kDa kilodalton - IR inverted repeat - ORF open reading frame - DALA -aminolevulinate     

Whole chloroplast genome comparison of rice,maize, and wheat: implications for chloroplast gene diversification and phylogeny of cereals

The fully sequenced chloroplast genomes of maize (subfamily Panicoideae), rice (subfamily Bambusoideae), and wheat (subfamily Pooideae) provide the unique opportunity to investigate the evolution of chloroplast genes and genomes in the grass family (Poaceae) by whole-genome comparison. Analyses of nucleotide sequence variations in 106 cereal chloroplast genes with tobacco sequences as the outgroup suggested that (1) most of the genic regions of the chloroplast genomes of maize, rice, and wheat have evolved at similar rates; (2) RNA genes have highly conservative evolutionary rates relative to the other genes; (3) photosynthetic genes have been under strong purifying selection; (4) between the three cereals, 14 genes which account for about 28% of the genic region have evolved with heterogeneous nucleotide substitution rates; and (5) rice genes tend to have evolved more slowly than the others at loci where rate heterogeneity exists. Although the mechanism that underlies chloroplast gene diversification is complex, our analyses identified variation in nonsynonymous substitution rates as a genetic force that generates heterogeneity, which is evidence of selection in chloroplast gene diversification at the intrafamilial level. Phylogenetic trees constructed with the variable nucleotide sites of the chloroplast genes place maize basal to the rice-wheat clade, revealing a close relationship between the Bambusoideae and Pooideae.     

Complete chloroplast genome sequences of Solanum bulbocastanum, Solanum lycopersicum and comparative analyses with other Solanaceae genomes

Despite the agricultural importance of both potato and tomato, very little is known about their chloroplast genomes. Analysis of the complete sequences of tomato, potato, tobacco, and Atropa chloroplast genomes reveals significant insertions and deletions within certain coding regions or regulatory sequences (e.g., deletion of repeated sequences within 16S rRNA, ycf2 or ribosomal binding sites in ycf2). RNA, photosynthesis, and atp synthase genes are the least divergent and the most divergent genes are clpP, cemA, ccsA, and matK. Repeat analyses identified 33–45 direct and inverted repeats ≥30 bp with a sequence identity of at least 90%; all but five of the repeats shared by all four Solanaceae genomes are located in the same genes or intergenic regions, suggesting a functional role. A comprehensive genome-wide analysis of all coding sequences and intergenic spacer regions was done for the first time in chloroplast genomes. Only four spacer regions are fully conserved (100% sequence identity) among all genomes; deletions or insertions within some intergenic spacer regions result in less than 25% sequence identity, underscoring the importance of choosing appropriate intergenic spacers for plastid transformation and providing valuable new information for phylogenetic utility of the chloroplast intergenic spacer regions. Comparison of coding sequences with expressed sequence tags showed considerable amount of variation, resulting in amino acid changes; none of the C-to-U conversions observed in potato and tomato were conserved in tobacco and Atropa. It is possible that there has been a loss of conserved editing sites in potato and tomato.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome.

Analysis of the mitochondrial DNA of a liverwort Marchantia polymorpha by electron microscopy and restriction endonuclease mapping indicated that the liverwort mitochondrial genome was a single circular molecule of about 184,400 base-pairs. We have determined the complete sequence of the liverwort mitochondrial DNA and detected 94 possible genes in the sequence of 186,608 base-pairs. These included genes for three species of ribosomal RNA, 29 genes for 27 species of transfer RNA and 30 open reading frames (ORFs) for functionally known proteins (16 ribosomal proteins, 3 subunits of H(+)-ATPase, 3 subunits of cytochrome c oxidase, apocytochrome b protein and 7 subunits of NADH ubiquinone oxidoreductase). Three ORFs showed similarity to ORFs of unknown function in the mitochondrial genomes of other organisms. Furthermore, 29 ORFs were predicted as possible genes by using the index of G + C content in first, second and third letters of codons (42.0 +/- 10.9%, 37.0 +/- 13.2% and 26.4 +/- 9.4%, respectively) obtained from the codon usages of identified liverwort genes. To date, 32 introns belonging to either group I or group II intron have been found in the coding regions of 17 genes including ribosomal RNA genes (rrn18 and rrn26), a transfer RNA gene (trnS) and a pseudogene (psi nad7). RNA editing was apparently lacking in liverwort mitochondria since the nucleotide sequences of the liverwort mitochondrial DNA were well-conserved at the DNA level.     

紫花苜蓿叶绿体基因组密码子偏好性分析

为分析紫花苜蓿叶绿体基因组密码子偏好性的使用模式,该文以紫花苜蓿叶绿体基因组中筛选到的49条蛋白质编码序列为研究对象,利用CodonW、CUSP、CHIPS、SPSS等软件对其密码子的使用模式和偏好性进行研究。结果表明:(1)紫花苜蓿叶绿体基因的第3位密码子的平均GC含量为26.44%,有效密码子数(ENC)在40.6～51.41之间,多数密码子的偏好性较弱。(2)相对同义密码子使用度(RSCU)分析发现,RSCU>1 的密码子数目有30个,以A、U结尾的有29个,说明了紫花苜蓿叶绿体基因组A或U出现的频率较高。(3)中性分析发现,GC3与 GC12的相关性不显著,表明密码子偏性主要受自然选择的影响; ENC-plot 分析发现一部分基因落在曲线的下方及周围,表明突变也影响了部分密码子偏性的形成。此外,有17个密码子被鉴定为紫花苜蓿叶绿体基因组的最优密码子。紫花苜蓿叶绿体基因组的密码子偏好性可能受自然选择和突变的共同作用。该研究将为紫花苜蓿叶绿体基因工程的开展和目标性状的遗传改良奠定基础。     

Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species

A detailed comparison was made of codon usage of chloroplast genes with their host (nuclear) genes in the four angiosperm speciesOryza sativa, Zea mays, Triticum aestivum andArabidopsis thaliana. The average GC content of the entire genes, and at the three codon positions individually, was higher in nuclear than in chloroplast genes, suggesting different genomic organization and mutation pressures in nuclear and chloroplast genes. The results of Nc-plots and neutrality plots suggested that nucleotide compositional constraint had a large contribution to codon usage bias of nuclear genes inO. sativa, Z. mays, andT. aestivum, whereas natural selection was likely to be playing a large role in codon usage bias in chloroplast genomes. Correspondence analysis and chi-test showed that regardless of the genomic environment (species) of the host, the codon usage pattern of chloroplast genes differed from nuclear genes of their host species by their AU-richness. All the chloroplast genomes have predominantly A- and/or U-ending codons, whereas nuclear genomes have G-, C- or U-ending codons as their optimal codons. These findings suggest that the chloroplast genome might display particular characteristics of codon usage that are different from its host nuclear genome. However, one feature common to both chloroplast and nuclear genomes in this study was that pyrimidines were found more frequently than purines at the synonymous codon position of optimal codons.     

Molecular Analysis of the Hot Spot Region Related to Length Mutations in Wheat Chloroplast Dnas. I. Nucleotide Divergence of Genes and Intergenic Spacer Regions Located in the Hot Spot Region

The nucleotide divergence of chloroplast DNAs around the hot spot region related to length mutation in Triticum (wheat) and Aegilops was analyzed. DNA sequences (ca. 4.5 kbp) of three chloroplast genome types of wheat complex were compared with one another and with the corresponding region of other grasses. The sequences region contained rbcL and psaI, two open reading frames, and a pseudogene, rpl23' (pseudogene for ribosomal protein L23) disrupted by AT-rich intergic spacer regions. The evolution of these genes in the closely related wheat complex is characterized by nonbiased nucleotide substitutions in terms of being synonymous/nonsynonymous, having A-T pressure transitions over transversions, and frequent changes at the third codon position, in contrast with the gene evolution among more distant plant groups where biased nucleotide substitutions have frequently occurred. The sequences of these genes had diverged almost in proportion to taxonomic distance. The sequence of the pseudogene rpl23' changed approximately two times faster than that of the coding region. Sequence comparison between the pseudogene and its protein-coding counterpart revealed different degrees of nucleotide homology in wheat, rice and maize, suggesting that the transposition timing of the pseudogene differed and/or that different rates of gene conversion operated on the pseudogene in the cpDNA of the three plant groups in Gramineae. The intergenic spacer regions diverged approximately ten times faster than the genes. The divergence of wheat from barley, and that from rice are estimated based on the nucleotide similarity to be 1.5, 10 and 40 million years, respectively.     

Gene distribution and isochore organization in the nuclear genome of plants.

The genomic distribution of 23 nuclear genes from three dicotyledons (pea, sunflower, tobacco) and five monocotyledons of the Gramineae family (barley, maize, rice, oat, wheat) was studied by localizing these genes in DNA fractions obtained by preparative centrifugation in Cs2SO4/BAMD density gradients. Each one of these genes (and of many other related genes and pseudogenes) was found to be located in DNA fragments (50-100 Kb in size) that were less than 1-2% GC apart from each other. This definitively demonstrates the existence of isochores in plant genomes, namely of compositionally homogeneous DNA regions at least 100-200 Kb in size. Moreover, the GC levels of the 23 coding sequences studied, of their first, second and third codon positions, and of the corresponding introns were found to be linearly correlated with the GC levels of the isochores harboring those genes. Compositional correlations displayed increasing slopes when going from second to first to third codon position with obvious effects on codon usage. Coding sequences for seed storage proteins and phytochrome of Gramineae deviate from the compositional correlations just described. Finally, CpG doublets of coding sequences were characterized by a shortage that decreased and vanished with increasing GC levels of the sequences. A number of these findings bear a striking similarity with results previously obtained for vertebrate genes.     

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.     

Rapid divergence of codon usage patterns within the rice genome

Background

Synonymous codon usage varies widely between genomes, and also between genes within genomes. Although there is now a large body of data on variations in codon usage, it is still not clear if the observed patterns reflect the effects of positive Darwinian selection acting at the level of translational efficiency or whether these patterns are due simply to the effects of mutational bias. In this study, we have included both intra-genomic and inter-genomic comparisons of codon usage. This allows us to distinguish more efficiently between the effects of nucleotide bias and translational selection.

Results

We show that there is an extreme degree of heterogeneity in codon usage patterns within the rice genome, and that this heterogeneity is highly correlated with differences in nucleotide content (particularly GC content) between the genes. In contrast to the situation observed within the rice genome, Arabidopsis genes show relatively little variation in both codon usage and nucleotide content. By exploiting a combination of intra-genomic and inter-genomic comparisons, we provide evidence that the differences in codon usage among the rice genes reflect a relatively rapid evolutionary increase in the GC content of some rice genes. We also noted that the degree of codon bias was negatively correlated with gene length.

Conclusion

Our results show that mutational bias can cause a dramatic evolutionary divergence in codon usage patterns within a period of approximately two hundred million years.The heterogeneity of codon usage patterns within the rice genome can be explained by a balance between genome-wide mutational biases and negative selection against these biased mutations. The strength of the negative selection is proportional to the length of the coding sequences. Our results indicate that the large variations in synonymous codon usage are not related to selection acting on the translational efficiency of synonymous codons.

     

Comparative analysis of codon usage patterns in chloroplast genomes of the Asteraceae family

Codon usage bias (CUB) is an important evolutionary feature in a genome and has been widely documented from prokaryotes to eukaryotes. However, the significance of CUB in the Asteraceae family has not been well understood, with no Asteraceae species having been analyzed for this characteristic. Here, we use bioinformatics approaches to comparatively analyze the general patterns and influencing factors of CUB in five Asteraceae chloroplast (cp) genomes. The results indicated that the five genomes had similar codon usage patterns, showing a strong bias towards a high representation of NNA and NNT codons. Neutrality analysis showed that these cp genomes had a narrow GC distribution and no significant correlation was observed between GC₁₂ and GC₃. Parity Rule 2 (PR2) plot analysis revealed that purines were used more frequently than pyrimidines. Effective number of codons (ENc)-plot analysis showed that most genes followed the parabolic line of trajectory, but several genes with low ENc values lying below the expected curve were also observed. Furthermore, correspondence analysis of relative synonymous codon usage (RSCU) yielded a first axis that explained only a partial amount of variation of codon usage. These findings suggested that both natural selection and mutational bias contributed to codon bias, while selection was the major force to shape the codon usage in these Asteraceae cp genomes. Our study, which is the first to investigate codon usage patterns in Asteraceae plastomes, will provide helpful information about codon distribution and variation in these species, and also shed light on the genetic and evolutionary mechanisms of codon biology within this family.     

Nucleotide sequence of the single ribosomal RNA operon of pea chloroplast DNA

The nucleotide sequence of an 8 kbp region of pea ( Pisum sativum L.) chloroplast DNA containing the rRNA operon and putative promoter sites has been determined and compared to the corresponding sequences from maize, tobacco and the liverwort Marchantia polymorpha . The chloroplast DNA species of all vascular plants investigated, with the exception of a few legumes including pea, and of Marchantia contain an inverted repeat with an rRNA operon. The pea rRNA operon is the first sequenced rRNA operon from a plant with only one copy of the rRNA genes per molecule of chloroplast DNA. The organization of the operon is the same as for maize, tobacco and Marchantia . i.e. tRNA-Val gene/16S rRNA gene/spacer with intron-containing genes for tRNA-Ile and tRNA-Ala/23S rRNA gene/4.5S rRNA gene/5S rRNA gene. Current evidence suggests that the tRNA-Val gene may not be contranscribed with the other genes. For pea 16S, 23S, 4.5S and 5S rRNA have 1488, 2813, 105 and 121 nucleotides, respectively. The homologies of the entire operon (the tRNA-Val gene - 5S rRNA region) to those from tobacco, maize and Marchantia are 88, 82 and 79%, respectively. The corresponding homologies for tobacco/maize, tobacco/ Marchantia and maize/ Marchantia have similar values. The 16S and 23S rRNA genes from pea are more than 90% homologous to those from the 3 other species. We conclude that the fact that pea only has one set of rRNA genes per molecule of chloroplast DNA is apparently not correlated with any significant difference between the pea operon and the rRNA operons from tobacco, maize and Marchantia .     

Synonymous codon usage in chloroplast genome of Coffea arabica

Synonymous codon usage of 53 protein coding genes in chloroplast genome of Coffea arabica was analyzed for the first time to find out the possible factors contributing codon bias. All preferred synonymous codons were found to use A/T ending codons as chloroplast genomes are rich in AT. No difference in preference for preferred codons was observed in any of the two strands, viz., leading and lagging strands. Complex correlations between total base compositions (A, T, G, C, GC) and silent base contents (A₃, T₃, G₃, C₃, GC₃) revealed that compositional constraints played crucial role in shaping the codon usage pattern of C. arabica chloroplast genome. ENC Vs GC₃ plot grouped majority of the analyzed genes on or just below the left side of the expected GC₃ curve indicating the influence of base compositional constraints in regulating codon usage. But some of the genes lie distantly below the continuous curve confirmed the influence of some other factors on the codon usage across those genes. Influence of compositional constraints was further confirmed by correspondence analysis as axis 1 and 3 had significant correlations with silent base contents. Correlation of ENC with axis 1, 4 and CAI with 1, 2 prognosticated the minor influence of selection in nature but exact separation of highly and lowly expressed genes could not be seen. From the present study, we concluded that mutational pressure combined with weak selection influenced the pattern of synonymous codon usage across the genes in the chloroplast genomes of C. arabica.